In this project, we will investigate the signaling and consequences of matrix metalloproteinase-9 (MMP-9) dysregulation in cells of the neurovascular unit (cerebral endothelial cells, astrocytes, neurons). Our overall hypothesis is that after stroke, oxidative stress and adhesion molecule signaling upregulates MMP-9 that degrades neurovascular matrix. This leads to not only blood-brain barrier leakage, but also disrupts cell-matrix interactions, thus triggering anoikis-like cell death in endothelium, astrocytes, and neurons. Angiotensin II, an inflammatory mediator, amplifies MMP-9. Treatment with statins and anti-adhesion agents downregulate MMP-9. To test this overall hypothesis, we will pursue 3 specific aims. In Aim 1, we will examine signaling pathways that upregulate MMP-9 using in vitro cultures of cerebral endothelial cells, astrocytes, and neurons. Responses to hypoxia/reoxygenation will be compared with responses to intracellular adhesion molecule (ICAM-1) receptor ligation. The roles of MAP kinase, STAT1, AP-1 and NFkB in MMP-9 regulaton will be assessed. In Aim 2, we will test the idea that MMP-9 upregulation triggers anoikis-like death in endothelial cells, astrocytes, and neurons. We will show that proteolytic degradation of extracellular matrix disrupts integrin linked kinase (ILK) and Akt cell survival signaling, thus activating caspase-mediated cell death. Pharmacologic inhibitors, RNA interference, and mutant ceils lacking MMP-9 or overexpressing the endogenous MMP inhibitor TIMP1 will be examined. In Aim 3, we will investigate the effects of modifying factors and therapy on MMP-9 levels in vitro (cell culture) and in vivo (mouse focal cerebral ischemia). Angiotensin II exposure in endothelial cultures and hypertensive renin/angiotensin overexpressing mice will be used to test for MMP-9 upregulation. Simvastatin and anti-ICAM-1 treatments will be tested to reduce MMP-9. In vivo optical and fluorescence imaging will be supported by the Scientific Core to measure changes in MMP activity, BBB permeability, and cerebral perfusion. Recent data from our lab and others established MMP-9 as a key protease that modifies neurovascular homeostasis. These proposed studies should provide novel insight into how MMP-9 becomes dysregulated and contributes to stroke pathophysiology.